Nano-Pulse Stimulation (NPS) is a non-thermal pulsed electric field modality that has been shown to have cancer therapeutic effects. Here we applied NPS treatment to the human papillomavirus type 16 (HPV 16)-transformed C3.43 mouse tumor cell model and showed that it is effective at eliminating primary tumors through the induction of immunogenic cell death while subsequently increasing the number of tumor-infiltrating lymphocytes within the tumor microenvironment. In vitro NPS treatment of C3.43 cells resulted in a doubling of activated caspase 3/7 along with the translocation of phosphatidylserine (PS) to the outer leaflet of the plasma membrane, indicating programmed cell death activity. Tumor-bearing mice receiving standard NPS treatment showed an initial decrease in tumor volume followed by clearing of tumors in most mice, and a significant increase in overall survival. Intra-tumor analysis of mice that were unable to clear tumors showed an inverse correlation between the number of tumor infiltrating lymphocytes and the size of the tumor. Approximately half of the mice that cleared established tumors were protected against tumor re-challenge on the opposite flank. Selective depletion of CD8⁺ T cells eliminated this protection, suggesting that NPS treatment induces an adaptive immune response generating CD8⁺ T cells that recognize tumor antigen(s) associated with the C3.43 tumor model. This method may be utilized in the future to not only ablate primary tumors, but also to induce an anti-tumor response driven by effector CD8⁺ T cells capable of protecting individuals from disease recurrence.

纳米脉冲刺激（NPS）是一种非热脉冲电场形式，已被证明具有癌症治疗作用。在这里，我们将NPS治疗应用于人乳头瘤病毒16型（HPV 16）转化的C3.43小鼠肿瘤细胞模型，并表明它可有效地通过诱导免疫原性细胞死亡来消除原发性肿瘤，并随后增加肿瘤浸润的数量肿瘤微环境内的淋巴细胞。体外NPS处理C3.43细胞导致活化的半胱天冬酶3/7翻倍，同时磷脂酰丝氨酸（PS）易位至质膜外小叶，表明程序性的细胞死亡活性。接受标准NPS治疗的荷瘤小鼠在大多数小鼠中显示出肿瘤体积的最初减少，随后肿瘤清除，并且总体存活率显着增加。对无法清除肿瘤的小鼠进行的肿瘤内分析显示，肿瘤浸润淋巴细胞的数量与肿瘤大小之间呈负相关。清除了已建立肿瘤的小鼠中大约有一半受到保护，可以防止对侧的肿瘤再次受到攻击。CD8 ^+的选择性耗竭T细胞消除了这种保护作用，表明NPS治疗可诱导产生适应性免疫应答，从而产生识别与C3.43肿瘤模型相关的肿瘤抗原的CD8 ⁺ T细胞。该方法将来可用于消融原发性肿瘤，还可以诱导由效应CD8 ⁺ T细胞驱动的抗肿瘤反应，该CD8 ⁺ T细胞能够保护个体免于疾病复发。